The objective of this research is the elucidation of the structural organization and functional significance in tumor cells of "homogeneously staining chromosomal regions" (HSRs) and extrachromosomal nuclear bodies termed double minutes (DM). Our working hypotheses are (1) that both of these karyotypic anomalies result from gene amplification, and (2) that they are directly related either to the expression of cell-specific traits, or to the tumorigenicity, of cells which contain them. As a model system, we will use two related sublines of a mouse steroid-secreting adrenocortical tumor cell line (Y1). One subline contains numerous DM (Y1-DM), while the other contains HSR-bearing marker chromosomes (Y1-HSR). We approach the research objectives by (1) physical isolation of DM, a defined component of the genome in these tumor cells, and (2) application of recombinant DNA technology to construct a library of DM-DNA fragments. This approach will allow the identification and characterization of transcriptional and translational products of DM and HSRs. The molecular nature and sequence complexity of DM-DNA will be defined by detailed restriction endonuclease analysis and nucleic acid reassociation techniques. Isolated DM-DNA fragments will be used as probes to examine the genomic organization and relative abundance of these sequences in Y1-DM, Y1-HSR, and normal cells. To identify that part of the genome from which the DM may have originated, appropriate somatic cell hybrids will be utilized to map these DNA sequences to specific mouse chromosomes.